Mechanism for Starch Granule Ghost Formation Deduced from

Jan 1, 2014 - We thank Dr. Fred Warren for the DSC data and fruitful discussions .... (22) Kasemsuwan, T.; Jane, J. L. Quantitative method for the sur...
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Mechanism for Starch Granule Ghost Formation Deduced from Structural and Enzyme Digestion Properties Bin Zhang, Sushil Dhital, Bernadine M. Flanagan, and Michael J. Gidley* Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia S Supporting Information *

ABSTRACT: After heating in excess water under little or no shear, starch granules do not dissolve completely but persist as highly swollen fragile forms, commonly termed granule “ghosts”. The macromolecular architecture of these ghosts has not been defined, despite their importance in determining characteristic properties of starches. In this study, amylase digestion of isolated granule ghosts from maize and potato starches is used as a probe to study the mechanism of ghost formation, through microstructural, mesoscopic, and molecular scale analyses of structure before and after digestion. Digestion profiles showed that neither integral nor surface proteins/lipids were crucial for control of either ghost digestion or integrity. On the basis of the molecular composition and conformation of enzyme-resistant fractions, it was concluded that the condensed polymeric surface structure of ghost particles is mainly composed of nonordered but entangled amylopectin (and some amylose) molecules, with limited reinforcement through partially ordered enzyme-resistant structures based on amylose (for maize starch; V-type order) or amylopectin (for potato starch; B-type order). The high level of branching and large molecular size of amylopectin is proposed to be the origin for the unusual stability of a solid structure based primarily on temporary entanglements. KEYWORDS: starch granule ghosts, gelatinization, α-amylase, enzyme-resistant fraction, glucan conformation, polymer entanglement

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INTRODUCTION Many agriculture and food biopolymers are assembled in an ordered or crystalline form in nature to confer stability and minimize hydration-driven swelling in the native environment. However, once these molecules or assemblies are heated above the relevant melting temperature, most become soluble, at least initially. Examples include many proteins and polysaccharides. Starch, however, is an exception, as when native semicrystalline granules are heated above their characteristic melting temperature under little or no shear, they swell and release some lower molecular weight components, but do not dissolve completely. Most applications of starch in food and industrial processes involve a heating step in the presence of water, which disrupts the ordered arrangement of polymers within granules (termed gelatinization). Gelatinized starches typically contain a mixture of solubilized polymers (mainly leached amylose and amylopectin molecules with low molecular weight) and residual granular structure (also termed (fragments of) granule “ghosts”).1 The importance of starch granule ghosts in determining the properties of cooked starches is often underestimated in studies that treat gelatinized starch as a fully dissolved polymer solution, like other biopolymer solutions such as agar and gelatin.2,3 The presence of the large (10−200 μm) macromolecular architecture of granule ghosts within an otherwise homogeneous polymer system can affect molecular processes, such as phase separation, and contribute to functional properties of starches that play important roles in texture and mouthfeel (e.g., viscosity, rheology, and tribology).2 Numerous studies of the structural changes associated with starch gelatinization have been reported,4−8 but the factors that contribute to the stability of granule ghost structures are still not clear. Various approaches have been used to understand the © XXXX American Chemical Society

robustness of granule ghosts, including selective extraction of surface nonpolysaccharide components, treatment of ghosts with protein-degrading enzymes, and the study of waxy maize mutants with variable (low) amylose contents.2,9 On the basis of these studies, it has been hypothesized that ghost formation is due to the cross-linking of amylose chains and/or long amylopectin branches (previously presumed to be by double helices), modulated by surface nonpolysaccharide components.2,9 However, direct experimental testing of this hypothesis has not been reported yet. Due to the lack of detectable ordered structure in granule ghosts1 (e.g., crystallinity from X-ray diffraction, helical order, or regularity of amylopectin clusters from small-angle Xray scattering), physical techniques used to study the intermolecular interaction of biopolymers, such as wide- and small-angle X-ray scattering, differential scanning calorimetry (DSC), 13C nuclear magnetic resonance (NMR), and Fourier transform infrared (FTIR) spectrometry, may provide limited insight into the mechanism of ghost formation and stabilization. We have recently shown, however, that there is a fraction of granule ghosts that is relatively resistant to enzymatic digestion.10 In this work we hypothesize that such enzyme-resistant fractions may be important in providing stabilizing structure to granule ghosts. We have therefore studied the susceptibility of granule ghosts toward enzymatic hydrolysis and investigated the macromolecular and ordered structures present in undigested residues as a probe of local structures that may be important in the formation and structure of ghosts. Received: October 18, 2013 Revised: December 18, 2013 Accepted: January 1, 2014

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by Schirmer et al.11 Aqueous suspensions of granule ghosts (0.5 mL) and 40 μL of aqueous Nile Blue solution (0.1 g/100 mL; N0766, SigmaAldrich) were transferred into 2 mL microcentrifuge tubes. After thorough mixing by repeated pipetting up and down, the stained solutions were incubated at 20 °C for 3 h. For confocal microscopic observation, the stained suspensions were dropped into the cavity of glass slides, sealed with a coverslip, and then observed using an LSM 700 confocal laser scanning microscope (CLSM, Zeiss). Excitation was at 639 nm with a diode laser operating at 2% of power capacity, and the emitted light was detected at an interval wavelength of 640−700 nm. Images of optical sections of granule ghosts were recorded with ZEN 2011 software (Zeiss). For SEM, ethanol-dried samples were thinly spread onto circular metal stubs covered with double-sided adhesive carbon tape and then platinum coated in a sputter coater. Images of the granule ghosts were acquired with a JEOL 6300 scanning electron microscope (JEOL Ltd., Tokyo, Japan) under an accelerating voltage of 5 kV. Particle Size Distribution. Particle size analysis was carried out using a Malvern Mastersizer Hydro 2000MU (Malvern Instruments Ltd., Malvern, UK) following the method of Dhital et al.12 A refractive index of 1.34 was used for granule ghost size calculation. The starch samples were added to circulating water until an obscuration of >10% was recorded. In Vitro Starch Digestion. In vitro starch digestion was performed with porcine pancreatic α-amylase, from the method described by Zhang, Dhital, and Gidley10 with slight modifications. Starch samples (50 mg, dry basis) were digested with 2.5 units of α-amylase in 30 mL of sodium acetate buffer (0.2 M, pH 6.0) in a shaking water bath at 37 °C. Aliquots (0.2 mL) were removed at different intervals, mixed with 1.0 mL of 95 °C water, and then boiled for 10 min to inactivate enzymes. The reducing sugar value was measured by the Nelson−Somogyi method.13 The maltose equivalent released (%) was calculated using the following equation (eq 1). Results were expressed as means with standard deviations of at least duplicate measurements.

Although there is some enzyme-resistant material present in granule ghosts formed under low shear cooking conditions from both maize and potato starches, high shear cooked starches are almost 100% digested, suggesting that granule ghosts are structurally different from high shear cooked starches.10 In this study, amylase digestion is used as a probe to investigate the structure of granule ghosts isolated from maize and potato, two commonly used commercial starches with different ghost profiles (e.g., size and integrity), as exemplars. On the basis of the concept that the enzyme-resistant fraction is important for maintaining ghost integrity, at least three hypotheses for the underlying structural basis for enzyme resistance can be proposed: (a) the multimicrometer structure of granule ghosts acts as a barrier to limit enzyme access; (b) cross-linking or dense entanglement of amylose and/or amylopectin branches limits enzyme action; or (c) proteins and lipids associated with the granule may contribute to either barrier or local cross-linking mechanisms. To test these hypotheses, in this study morphological parameters of ghosts and digested remnants have been studied with different microscopic techniques, the molecular structure evolution during amylase digestion of granule ghosts has been monitored by 1H NMR spectroscopy and size exclusion chromatography (SEC), and the conformation of polymers within ghosts and enzyme-resistant residues has been determined by 13C CP/MAS NMR. On the basis of the data obtained, mechanisms for starch granule ghost formation and stability are discussed.

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EXPERIMENTAL PROCEDURES

Materials. Maize starch (MS) was purchased from Penford Australia Ltd. (Lane Cove, NSW, Australia), and potato starch (PS) was from Sigma-Aldrich. (St. Louis, MO, USA). Porcine pancreatic α-amylase (A3176, activity = 23 unit/mg) and other chemicals were obtained from Sigma-Aldrich. Depletion of Proteins and Lipids from Granule Surfaces. Treatment of starch granule slurries (20% w/v) with sodium dodecyl sulfate (SDS, 2% w/v) at 20 °C for 30 min was used to extract surface proteins and lipids.9 Extracted granules were isolated by centrifugation and washed with >10 volumes of cold deionized water until no foaming was observed in the washings. Despite the extensive washing, some SDS was retained by the granules as shown by the increase in sodium and sulfur contents after treatment (Supporting Information, Table S1). Preparation of Cooked Starch and Granule Ghosts. Starch granule aqueous slurry (10 mL, 0.5% w/v) in a 50 mL centrifuge tube (with a magnetic stirrer, 3 mm × 8 mm) was heated to 100 °C on a hot plate stirrer at a stirring rate of 1500 rpm for 30 min (“high shear cooking”) and designated “cooked starch”. Granule ghosts were isolated from maize and potato starches following an adaptation of a method reported previously.1,2 Starch (200 mg) was suspended in a small amount of cold water and then poured into hot water (95 °C, 40 mL). The dilute suspension (0.5% w/v starch) was kept at 95 °C for 30 min at a low stirring rate (250 rpm, “low shear cooking”) and then centrifuged (30 °C, 2000g for 15 min). The supernatant was removed, and the spun ghosts were washed twice by resuspension in hot water (90 °C) with gentle manual stirring followed by centrifugation. Ghost yield was defined as the weight ratio of freeze-dried pellet to initial starch. The washed ghosts were finally resuspended in excess water for microscopy and in vitro digestion. For SEC, NMR, and scanning electron microscopy (SEM) measurements, starch samples were precipitated by absolute ethanol and dried under pressured nitrogen gas overnight. Microscopy. Light microscopy was performed using a Zeiss Axio microscope (Oberkochen, Germany). One drop of fresh granule ghost suspension was placed on a glass slide and stained with 2% iodine solution, and the images were recorded by AxioCam ERc5s camera (Zeiss). For confocal microscopy, the staining procedure of fresh granule ghosts with Nile Blue was adapted from the method described

maltose equivalent released (%) total wt of equivalent maltose in supernatant = × 100 dry wt of starch

(1)

Size Exclusion Chromatography. The fully branched and debranched size distribution of starch molecules during the course of in vitro digestion was obtained from an SEC system (Agilent 1100, Agilent Technologies, Waldbronn, Germany) equipped with a refractive index detector (RID-10A, Shimadzu, Kyoto, Japan) following the methods of Cave et al.14 and Hasjim et al.15 SEC samples were prepared following the method described elsewhere15 and then injected into the following series of columns: precolumn, Gram30, Gram3000 (PSS, Mainz, Germany) for the fully branched distribution, and precolumn, Gram100, Gram 1000 for debranched distribution. The molecular size distribution data were plotted as SEC weight distribution, w(log Vh), against the hydrodynamic radius (Rh/nm). For linear polymers of uniform geometry, the size and molecular weight (or equivalently the degree of polymerization, DP) are uniquely related, and hence the size distribution can be processed to a molecular weight distribution using the Mark−Houwink equation.14,16 SEC calibration was performed using pullulan standards with molecular weights ranging from 342 to 1.66 × 106 Da (PSS, Mainz, Germany). The standards were dissolved in the SEC eluent and injected into the branched and debranched SEC setups to provide universal calibration curves to relate elution volume with Rh. Because of the calibration range, the Rh values above the upper limit of the standards available (∼50 nm) are only semiquantitative. The amylose content can also be calculated as the ratio of the area under the curve (AUC) of the debranched SEC distribution curves for the larger branches to the total AUC for all branches.17 The amylose contents of maize and potato starches determined in this way were 23.3 ± 1.0 and 18.3 ± 0.4% respectively. 1 H Nuclear Magnetic Resonance Spectroscopy. The degree of branching (DB) of starch molecules during the course of in vitro digestion was measured on a Bruker Avance NMR spectrometer (Bruker Biospin, Rheinstetten, Germany), operating at a Larmor B

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Figure 1. Light micrographs and particle size distribution of fresh granule ghosts (A, maize, scale bar = 20 μm; B, potato, scale bar = 100 μm) with/ without SDS pretreatment or high shear force (1, without any treatment; 2, with SDS pretreatment; 3, with high shear force treatment after ghost formation). Insets in panels A1 and B1 are confocal microscopy images after staining with Nile Blue. 13 C CP/MAS Nuclear Magnetic Resonance Spectroscopy. Starch samples were examined using a solid-state 13C CP/MAS NMR spectrometer (Bruker MSL-300, Rheinstetten, Germany) at a 13C frequency of 75.46 MHz. Approximately 200 mg of sample was packed in a 4 mm diameter, cylindrical, partially stabilized zirconium oxide rotor with a KelF end-cap. The rotor was spun at 5 kHz at the magic angle (54.7°). The 90° pulse width was 5 μs, and a contact time of 1 ms was used for all samples with a recycle delay of 3 s. The spectral width was 38 kHz; acquisition time, 50 ms; time domain points, 2L; transform size, 4K; and line broadening, 50 Hz. At least 2400 scans were accumulated for each spectrum. In the case of the 3 h digestion residues of maize and potato ghosts, it was necessary to collect 20000 scans, as only 50 mg was available. Spectra were referenced to external adamantine and were

frequency of 750 MHz for 1H, equipped with a TXI5z probe following the method of Tizzotti et al.18 1H NMR spectra were recorded at 333 K with an 8 μs 90° pulse, a repetition time of 15.98 s (composed of an acquisition time of 3.98 s and a relaxation delay of 12 s), and 128 scans. The addition of a very low amount of TFA-d1 to the medium causes the exchangeable protons of the starch hydroxyl groups and of the residual water to shift to higher frequency, leading to clear and well-defined 1H NMR spectra.18 DB is obtained using eq 2

DB (%) =

Iα ‐ (1,6) Iα ‐ (1,4) + Iα ‐ (1,6)

× 100 (2)

where Iα‑(1,4) and Iα‑(1,6) are the H NMR integrals of internal α-(1,4) and α-(1,6) linkages at ∼5.12 and ∼4.78 ppm, respectively. 1

C

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Figure 2. SEM micrographs of ethanol-precipitated granule ghosts (A−D, maize; E and F, potato).

typically with a particle size between 15 and 35 μm (Figure 1A1). In contrast, potato ghosts have a wide range of particle sizes (typically 50−150 μm) with more ellipsoidal shapes and significant amounts of granule fragments (Figure 1B1). Expansion is so extensive that the resultant potato ghosts (ca. 4 times expansion in original diameter, i.e., ca. 64-fold increase in granular volume) are more fragile and sensitive to shear force, compared with maize ghosts (increasing only ca. 2 times in diameter) even under the low shear conditions used in ghost preparation. This is not surprising because potato starch contains an appreciable amount of phosphate monoester groups,22 which are negatively charged and linked to potato amylopectin molecules. The resulting charge repulsion effect helps to untangle the individual branches and extends the degree of granule swelling.23 Maize ghosts frequently appeared to have more localized regions of folds and wrinkles (Figure 1A1,B1, insets), which may trap undispersed starch polymers.3 The morphology of granule ghosts was also investigated using CLSM after being stained by Nile Blue, a water-soluble basic oxazine dye, which is one of the most suitable fluorescent staining agents for both granular and gelatinized starches in the absence of

analyzed by resolving the spectra into ordered and amorphous subspectra and calculating the relative areas as described previously.19 When percentage order is calculated, freeze-dried starches are compared with freeze-dried amorphous standards, and samples precipitated with ethanol are compared with ethanol-precipitated amorphous standards.

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RESULTS AND DISCUSSION Microscopic Structure of Granule Ghosts. Fresh granule ghosts from maize and potato starches were isolated by centrifugation following the method reported by Debet and Gidley.2 This method ensures full gelatinization and maximum swelling (for most normal starches) when starch is heated at 95 °C for 30 min in excess water. The risks of retrogradation from leached amylose and shear degradation are minimized by the low starch concentration (10 nm, DP >500) (Figures 4C and 5C), suggesting that α-amylase tends to cleave amylopectin molecules into clusters (which provide a barrier to enzyme access and hence prevent preferential digestion of intercluster amylopectin molecules32) and that digestion within clusters occurs at random. At the beginning of digestion (20 min), there was a gradual degradation of starch molecules as the peaks of both whole and debranched distributions were slightly shifted to lower Rh regions (Figures 4 and 5D,F,I,J). In addition, it is noteworthy that the debranched chain distributions of 3-hdigested maize ghosts had bimodal peaks, whereas those from potato ghosts showed only one peak at Rh ∼1.5 nm with a shoulder that diminishes in size without changing shape (Figures 4E and 5E). The bimodal peaks of maize ghost residues are designated highly branched α-limit dextrin (peak observed at Rh ∼1 nm) and longer chain polymers (Rh peak ∼3.4 nm) respectively, which are consistent with their whole molecule distributions and DB values (Figures 4E,J; Table 1). Clusters of branching points within starch molecules will slow the amylase action because of steric hindrance. It is noteworthy that some long-chain polymers (DP >100) survived, presumably from degraded amylose in original maize ghost particles. Cuevas et al.33 also found that the hot-water-insoluble fractions in waxy rice starch contained DP ≥100 chains, which were absent in the hotwater-soluble fractions. However, these long-chain components were not found for the potato ghosts with the same digestion time and similar digestion extents (Figure 3), in agreement with the higher DB values, compared with that of maize residues (Table 1). Furthermore, the α-limit dextrin peak indicates that whole amylopectin present in the ghosts is reduced in size by αamylase to ∼2.5 nm remnant molecules, which is consistent with previous results.15,31 Conformation of Ghost Residues after Enzyme Digestion. The macromolecular architecture of an apparently amorphous matrix such as granule ghosts is critical to their formation and stability. For starch granules, Biliaderis et al.34 proposed a three-phase model incorporating two distinct types of amorphous materials, that is, nonordered intercrystalline and bulk amorphous matrix, together with the crystalline domains of amylopectin clusters, accounting for order−disorder phase transitions of starch gelatinization. However, there is no reason to suppose that the same amorphous structures exist after gelatinization. As a probe of molecular conformation, molecular order (single helices, double helices) at short distance scale solidstate CP/MAS NMR spectra were recorded before and after enzyme digestion. The 13C CP/MAS NMR spectra of maize and potato ghosts prepared by freeze-drying or ethanol precipitation produce distinct NMR spectra (Figure 6A). Regardless of the botanical origin of starch, when the starches from the two drying methods are intensity matched at 84 ppm (Figure 6A), there is a difference in intensity between 92 and 100 ppm. By instead matching the intensity of a freeze-dried ghost spectrum, and an ethanolprecipitated ghost spectrum in the 92−100 ppm region, and then

detection limit of measurement techniques or the polymer chains are just physically entangled without any fixed conformation in the skins of ghosts. Molecular Structures Present in Granule Ghosts and Their Evolution during Amylase Digestion. Molecular size distribution of whole maize and potato starch molecules (Figures 4F and 5F, respectively) and chain length distributions of enzymatically debranched maize and potato starch samples (Figures 4A and 5A) were characterized using SEC. The fully branched SEC weight distribution of the whole molecules of the two starches showed an amylopectin peak (Rh between 40 and 300 nm, peak Rh = ∼100 nm) and a shoulder for amylose molecules stretching from Rh of 1 to 40 nm. However, some hybrid components could also be present, such as molecules that are highly branched like amylopectin but with molecular size similar to that of amylose and also amylopectin molecules with extra-long branches.30 The debranched SEC weight distribution can be empirically divided into two regions representing amylopectin branches (single-lamella branches, peak Rh ∼1.5 nm or DP ∼12; trans-lamella branches, peak Rh ∼2.5 nm or DP ∼50) and amylose branches (Rh ∼3.5−80 nm, DP ∼100− 30000).15,31 Amylose molecules are typically smaller than amylopectin and tend to leach out from granules during swelling, as shown by amylose contents of maize (8.4%) and potato (6.7%) ghosts, which are much lower than the starting starches (Figures 4B and 5B). Comparison of Figures 4F/5F with Figures 4G/5G shows that ghost formation is accompanied by loss of the lower molecular size molecules, again consistent with the amylose content results. During ghost formation, 15.1 and 38.8% of amylopectin (calculated from the amylose content of both granules and ghosts and the isolated yields of ghosts, see the Supporting Information) is leached out of maize and potato granules, respectively, suggesting that either a proportion of ghosts do not survive the cooking process or that amylopectin molecules are leached alongside amylose. Despite differences in particle size and robustness, ghosts from maize and potato starch have similar fully branched and debranched molecular size distributions (Figures 4B,G and 5B,G). The debranched and fully branched SEC weight distributions of maize and potato ghosts and their evolution during amylase digestion are shown in Figures 4 and 5, and DB values calculated from 1H NMR spectra are summarized in Table 1. DB values increase with digestion time, consistent with branched residues being less susceptible to digestion than nonbranched residues, and potato DB values reach higher levels, suggesting a difference in the relative enzyme susceptibility of nonbranched residues between maize and potato ghosts. The size distribution of whole starch molecules reveals that both amylose and amylopectin were Table 1. DB Values (Number of Branching Points as a Percentage of the Total Number of Glucosidic Linkages) of Maize and Potato Ghosts and Their Evolution during Amylase Digestion samplea

DB (%)

samplea

DB (%)

MS MS-G MS-G-20 MS-G-60 MS-G-180

2.81 3.02 7.81 8.57 7.35

PS PS-G PS-G-20 PS-G-60 PS-G-180

2.11 2.17 4.82 12.75 11.95

a

MS, maize starch; PS, potato starch; G, granule ghosts; 20, 60, and 180 represent hydrolysis for 20, 60, and 180 min, respectively. H

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Figure 6. 13C NMR spectra: (A) maize and potato ghosts with freeze-drying (FD) or ethanol-drying (ED); (B) maize ghosts with freeze- or ethanoldrying; (C) digestion residues from maize and potato ghosts. Separation of 13C NMR spectra into ordered subspectra: (D) digestion residues from maize and potato ghosts. MS, maize starch; PS, potato starch; G-180, granule ghost hydrolysis for 180 min.

subtracting the two spectra, a spectrum of V-type helices is revealed (Figure 6B). Spectra of all ethanol-precipitated ghosts clearly indicated the presence of V-type helices with peaks for C1 at 102.9 ppm and C4 at 81.5 ppm.35 After 3 h of enzyme digestion, the resistant material was recovered by ethanol precipitation to separate it from the large amount of low molecular weight digestion products. NMR spectra (Figure 6C) therefore showed intensity consistent with V-type helices with peaks at 102.9 and 81.5 ppm. The ordered subspectra (Figure 6D) were obtained by matching the intensity at 84 ppm of the 3 h digestion residues and an ethanol-dried amorphous maize starch standard and subtracting one from the other. Although there is no obvious order present when the total spectrum of 3-hdigested potato ghost is compared with the amorphous standard, upon subtraction of the two spectra it becomes apparent that a small amount of B-type double helices (13 ± 2%) is found for the potato ghost digestion residue with peaks at 100.3 and 99.5 ppm.35−38 In contrast, the spectrum of the 3 h digestion residue from maize ghosts shows more V-type (19 ± 2%) than the standard and the starting ghost material. As shown in Figure 6A,B, ghosts (residues) isolated by ethanol precipitation have some V-type character. Consistent with this, digestion residues from both maize and potato starch ghosts have a signal for ethanol in the NMR spectrum (Figure 6C, 17 ppm). However, there are clearly also signals for lipids in the 3 h digestion residue from maize ghosts (Figure 6C, 33 and 23 ppm), and similar levels

of ethanol peaks for both maize and potato 3 h digestion residues (Figure 6C, 17 ppm) can be detected.39 We therefore propose that the enzyme-resistant V-type helices found in the digestion residue from maize ghosts are due to amylose−lipid complexes either present in the starting granule or formed during gelatinization and/or digestion processes.40 Proposed Mechanism for Granule Ghost Formation. At the early stage of heat/moisture-induced starch swelling, even at lower temperatures than those required for crystallite melting, amylose and a small amount of amylopectin molecules can leach out of granules.41 Crystallite melting,4 driven by double-helix dissociation,5 causes granule swelling essentially by replacing hydrogen bonds between amylopectin chains with hydrogen bonds to water molecules,42 thereby allowing swelling of high molecular weight amylopectin. Swelling behavior is primarily a property of amylopectin,43 a main molecular component of ghosts, whereas amylose acts both as a diluent and as an inhibitor of swelling, especially in the presence of lipids/proteins, which can form insoluble complexes with amylose.42,44 An intact amylopectin surface is thought to form a continuous layer surrounding internal starch granule components.3,28 At certain critical stress points of granules, swelling eventually ruptures the envelope (e.g., Figure 2F), presumably by breakdown of the molecular interactions that holds the granule surface together. On the basis of the high level of enrichment of high molecular size amylopectin, we suggest that molecular interaction between I

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can result in an effectively permanent multimicrometer structure. Small amounts of amylose molecules may also be important to hold amylopectin molecules together by physical entanglements (e.g., maize and potato ghosts) and strengthen the cross-linking entanglements between amylopectin molecules. For example, starches from waxy maize lines with variable low levels of amylose formed granule ghosts with yield and integrity dependent on amylose content.2 Surface proteins and lipids can also affect ghost properties. For those starches (e.g., wheat and maize) that have restricted swelling due to surface proteins/lipids,9 ghosts prepared from SDS-treated granules show greater expansion due to the removal of constraining proteins and lipids, leading to a greater extent of swelling before sufficient cross-linking interactions occur to prevent dissolution. In conclusion, the high molecular weight and highly branched primary structure of amylopectin will lead to many dynamic entanglements, which in combination seem to be sufficient to retain a stable ghost structure. The formation of ghosts as a consequence of extensive swelling of granules following gelatinization suggests that entanglements are formed during the swelling process, explaining why stable hydrated nonordered starch structures starting from preisolated amylopectin have not been reported. The proposed mechanism for granule ghost formation not only confirms that double helices are not necessary to strengthen ghost structure but also raises the question as to how enzyme-resistant fractions could be formed from an essentially amorphous (entangled) starch matrix. Crystallinity alone does not always lead to an increase in enzyme resistance, and almost amorphous high-amylose starches can provide high yields of resistant fraction, although it has generally been accepted that crystallinity must play some role in determining enzyme resistance.46−48

amylopectin units is responsible for the surface integrity of ghost particles. It has previously been shown by both small-angle and wide-angle X-ray scattering that there is no detectable crystalline or lamellar order in barley starch ghosts.1 This lack of detectable organizational structure was further extended in this study through both DSC, which showed no detectable melting endotherms (Supporting Information, Figure S2), and solid state 13C NMR, which showed only amorphous features for both maize and potato freeze-dried ghost samples (Figure 6A). However, it is possible that a small amount of cross-linking through, for example, double helices could be present below detection levels of the X-ray, NMR, and DSC techniques. The finding that there is a small amount of an enzyme-resistant fraction in maize (3.8 ± 0.3%, calculated by weight of 3 h digestion residue) and potato (1.5 ± 0.4%) starch ghosts suggests the possibility that this fraction provides a cross-linking or other stabilizing function in otherwise amorphous granule ghost structures. From NMR (Figure 6) and SEC (Figures 4 and 5) results, the enzyme-resistant fraction of maize ghosts is enriched in amylose with 19% of the sample in a lipid-complexed single-helical form, and the enzyme-resistant portion of potato ghosts is enriched in amylopectin branches/clusters with 13% of the sample present as B-type double helices. In the case of maize, no evidence has been found for the presence of any potentially cross-linking double helices within ghosts, suggesting that double helices are not necessary to provide structure to ghost particles. Although the Vtype helices from amylose−lipid complexes are not responsible for cross-linking starch chains as they involve only a single starch chain, they may help to rigidify segments of amylose within maize ghosts. Potato ghosts are more highly swollen and more sensitive to shear than maize ghosts, probably due to the repulsion effect from negatively charged phosphate groups during swelling. Despite similar amylose contents and enzyme digestibility to maize starch ghosts, the enzyme-resistant fraction from potato ghosts is qualitatively different, being based on amylopectin and containing at least some double helices. Normally, B-type double helices would be expected to be formed more readily from amylose and thereby potentially act to cross-link starch polymers. SEC data (Figure 5E,J) for potato ghost enzyme-resistant residue, however, show that it contains predominantly amylopectin, consistent with largely intra-amylopectin double helices formed from adjacent branches, although cross-linking between amylopectin molecules cannot be ruled out. The presence of double helices in enzyme-resistant fractions of potato but not maize starch ghosts is consistent with the fact that the chain length distribution of amylopectin from potato is longer than that of maize (Figures 4A and 5A), and the absence of Vtype helices is consistent with the much lower lipid content of potato compared to maize starch.45 However, it should be noted that the amount of double helices is low (∼13% calculated for the 3 h digestion residue, corresponding to ∼0.2% based on total starch ghost explaining why it cannot be detected in nonenzyme-treated ghosts) and, therefore, not likely to be a primary mechanism for ghost formation. On the basis of the lack of detectable ordered structure in intact ghosts, and the low level of order within the small fraction of ghosts that are enzyme resistant, it is apparent that the stable structure of ghosts is derived primarily from simple entanglement of nonordered polymers, particularly amylopectin. The highly branched primary structure and very high molecular size of amylopectin (Figure 5) mean that each molecule can be involved in a large number of temporary entanglements, the sum of which

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ASSOCIATED CONTENT

* Supporting Information S

Methods and results of protein contents and mineral compositions, the influence of maltose on the kinetics of αamylase digestion, the DSC thermograms of ghost and their digestion residue samples. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(M.J.G.) Phone: +61 7 3365 2145. Fax: +61 7 3365 1177. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Fred Warren for the DSC data and fruitful discussions and Dr. Jovin Hasjim and Sarah Chung for their technical assistance with SEC measurement. We also acknowledge the facilities and the technical assistance of Dr. Kim Sewell, Australian Microscopy and Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, The University of Queensland.

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ABBREVIATIONS USED AUC, area under the curve; CLSM, confocal laser scanning microscopy; DB, degree of branching; DP, degree of polymerization; DSC, differential scanning calorimetry; FTIR, Fourier transform infrared; MS, maize starch; NMR, nuclear magnetic J

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resonance; PS, potato starch; Rh, hydrodynamic radius; SDS, sodium dodecyl sulfate; SEC, size exclusion chromatography; SEM, scanning electron microscopy

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